Cj. Herscher et al., PRE-STEADY-STATE KINETIC-STUDY OF THE EFFECTS OF K-MEMBRANE CA2+-ATPASE( ON THE PARTIAL REACTIONS OF THE CATALYTIC CYCLE OF THE PLASMA), Biochemical journal, 315, 1996, pp. 673-677
The effects of 100 mM K+ on the partial reactions that take place duri
ng ATP hydrolysis by the calcium ion-dependent ATPase from plasma memb
rane (PM-Ca2+-ATPase) were studied at 37 degrees C on fragmented intac
t membranes from pig red cells by means of a rapid chemical quenching
technique. At 10 mu M [gamma-P-32]ATP plus non-limiting concentrations
of Ca2+ and Mg2+, K+ increased the k(app) of formation by 140 %, to 8
4 +/- 11 s(-1) and the steady-state level of phosphoenzyme (EP) by 25
%, to 3.4 +/- 0.17 pmol/mg of protein. If added together with [gamma-P
-32]ATP at the beginning of phosphorylation, K+ was much less effectiv
e than if added earlier, indicating that it did not act on the phospho
rylation reaction. Measurements of the E(2) --> E(1) transition by pho
sphorylation showed that in medium with Ca2+ and Mg2+, K+ increased th
e k(app) of the transition by 55 %, to 14 +/- 3 s(-1) and the apparent
concentration of E(1) by 45 %, suggesting that this may be the cause
of the increased rate of phosphorylation observed in enzyme preincubat
ed with K+. The presence of K+ did not change the slow decay of EP wit
hout Mg2+ but activated the decay of EP made with Mg2+, increasing its
k(app) by 60 %, to 91 +/- 12 s(-1). In contrast with observations mad
e during phosphorylation, if added at the beginning of dephosphorylati
on K+ was fully effective in favouring decomposition of EP made in med
ium containing no K+. In the presence of either 3 mM ATP or 3 mM ATP p
lus calmodulin, which activate hydrolysis of CaE(2)P, the effect of K on dephosphorylation was conserved. Because the sites for K+ are intr
acellular and the concentration of K+ in normal red cells is above 100
mM, the effects described here must be taken into account to describe
the catalytic cycle of the PM-Ca2+-ATPase under physiological conditi
ons.